Editor’s Highlight: Pregnancy Alters Aflatoxin B1
Metabolism and Increases DNA Damage in Mouse Liver
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Sriwattanapong, Kanokwan et al. “Editor’s Highlight: Pregnancy
Alters Aflatoxin B1 Metabolism and Increases DNA Damage in
Mouse Liver.” Toxicological Sciences 160, 1 (August 2017): 173–179
© 2017 The Author
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http://dx.doi.org/10.1093/TOXSCI/KFX171
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Oxford University Press (OUP)
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Final published version
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http://hdl.handle.net/1721.1/117600
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Creative Commons Attribution-NonCommercial 4.0 International
Pregnancy Alters Aflatoxin B
1
Metabolism and
Increases DNA Damage in Mouse Liver
Kanokwan Sriwattanapong,*
,†,1Stephen L. Slocum,*
,1Supawadee
Chawanthayatham,* Bogdan I. Fedeles,* Patricia A. Egner,
‡John D. Groopman,
‡Jutamaad Satayavivad,
†Robert G. Croy,* and
John M. Essigmann*
,2*Departments of Biological Engineering and Chemistry, and Center for Environmental Health Sciences,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
†Graduate Program in Environmental
Toxicology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand; and
‡Department of Environmental
Health Sciences, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
1These authors contributed equally to this study.
2To whom correspondence should be addressed. Fax: (617) 253-5445. E-mail: jessig@mit.edu.
ABSTRACT
Pregnancy is a complex physiological state, in which the metabolism of endogenous as well as exogenous agents is ostensibly altered. One exogenous agent of concern is the hepatocarcinogen aflatoxin B1(AFB1), a foodborne fungal toxin, that requires phase I metabolic oxidation for conversion to its toxic and carcinogenic form, the AFB1-8,9-exo-epoxide. The epoxide interacts with cellular targets causing toxicity and cell death; these targets include the covalent modification of DNA leading to mutations that can initiate malignant transformation. The main detoxification pathway of the AFB1-epoxide involves phase II metabolic enzymes including the glutathione-S-transferase (GST) family. Pregnancy can
modulate both phase I and II metabolism and alter the biological potency of AFB1. The present work investigated the impact of pregnancy on AFB1exposure in mice. A single IP dose of 6 mg/kg AFB1was administered to pregnant C57BL/6 J mice at gestation day 14 and matched non-pregnant controls. Pregnant mice accumulated 2-fold higher AFB1-N7-guanine DNA adducts in the liver when compared with nonpregnant controls 6 h post-exposure. Enhanced DNA adduct formation in pregnant animals paralleled elevated hepatic protein expression of mouse CYP1A2 and mouse homologs of human CYP3A4, phase I enzymes capable of bioactivating AFB1. Although phase II enzymes GSTA1/2 showed decreased protein expression, GSTA3, the primary enzymatic protection against the AFB1-epoxide, was unaffected at the protein level. Taken together, our results reveal that pregnancy may constitute a critical window of susceptibility for maternal health, and provide insight into the biochemical factors that could explain the underlying risks.
Key words: Aflatoxin B1; DNA adducts; early life exposure; maternal exposure; maternal fetal health axis.
In excess of 750 000 people die of hepatocellular carcinoma (HCC) each year, making HCC the second leading cause of can-cer deaths worldwide (Ferlay et al., 2013). Major risk factors for HCC include chronic infection with hepatitis B or C viruses and dietary exposures to aflatoxin B1(AFB1) (WHO, 2013;Wild and
Gong, 2010). Individual exposure to AFB1or HBV results in a hu-man liver cancer risk enhancement of 3.4- and 7-fold, respec-tively (Kensler et al., 2011;Qian et al., 1994); that risk rises to 60-fold when both factors are present concurrently. The International Agency for Research on Cancer classifies AFB1as a
VCThe Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
173
doi: 10.1093/toxsci/kfx171
Advance Access Publication Date: August 24, 2017 Research Article
Group 1 agent—known human carcinogen—based on evidence from studies of AFB1-treated animals, epidemiological studies in exposed populations and studies involving functional bio-markers that mechanistically predict disease (IARC, 1993).
The aflatoxins are a group of mycotoxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus (Moss, 1998) that readily contaminate many staple foods including nuts, corn and cereals. Contamination occurs most frequently in developing countries with tropical and subtropical climates, where food stor-age and quality control practices are inadequate (Strosnider et al., 2006). Up to 4.5 billion people are believed to be at risk of afla-toxin exposure (Liu and Wu, 2010). Among the aflatoxins, AFB1is the most common and most toxic variant (Edwards et al., 1971;
Wogan et al., 1971). Its primary mechanism of toxicity and carci-nogenicity is the bioactivation of AFB1by phase I enzymes (ie, cytochromes P450) to a highly reactive epoxide (AFB1-8,9-exo-ep-oxide) that can covalently modify cellular components, including DNA. Epidemiological studies have identified AFB1-DNA adducts as biomarkers of AFB1exposure, and predictors of HCC risk in humans (Egner et al., 2006;Groopman et al., 2008).
The B6C3F1 mouse remains a versatile and commonly deployed model for AFB1 exposure and carcinogenicity (Vesselinovitch et al., 1972). During the neonatal period, admin-istration of a single dose of AFB1results in 90–100% incidence of liver cancer in male B6C3F1 mice, whereas females have a 10% incidence (Chawanthayatham et al.,2015, 2017;Vesselinovitch et al., 1972;Wattanawaraporn et al., 2012). The biological basis for the differential AFB1 sensitivity between sexes is not completely understood. Previous work shows that both male and female neonates form similar levels of the strongly muta-genic AFB1-guanine adducts measured 6 h post-toxin adminis-tration, with both sexes demonstrating similar levels of characteristic AFB1 mutations at 10 weeks after dosing (Chawanthayatham et al., 2015;Wattanawaraporn et al., 2012;
Woo et al., 2011). Similarly, the 10-week mutation frequencies of male and female mice treated in utero are identical (Chawanthayatham et al., 2015). These data suggest that post-pubescent sex-specific biochemical factors may determine the differential sensitivity of males to AFB1-induced HCC.
In humans, AFB1 is metabolized by cytochromes P450 3A4 (CYP3A4) and 1A2 (CYP1A2), forming the AFB1-epoxide (Gallagher et al., 1994). Reaction of the epoxide with DNA produces a charac-teristic biomarker, the AFB1-N7-guanine adduct (Bennett et al.,
1981;Croy and Wogan, 1981;Egner et al., 2006;Groopman et al., 1981). The AFB1-epoxide can be detoxified by the glutathione-S-transferases (GSTs), via conjugation to glutathione (GSH) (Figure 1) (Dohnal et al., 2014). The metabolic activation of AFB1has not been fully elucidated in mice; specifically, studies that examine the particular P450 enzymes involved in bioactivation are lack-ing. Mice express an orthologue of human Cyp1A2, which osten-sibly can oxidize AFB1 into the AFB1-epoxide, but they lack a direct orthologue of Cyp3A4. However, mice do have a plethora of potential functional homologs among their many metabolic cytochromes (Nelson et al., 2004). Indeed, a phylogenetic analysis reveals several members of the mouse CYP3A family that are closely related to human CYP3A4 (Supplementary Figure 1). The murine detoxification pathways for aflatoxin are better under-stood, with mouse GSTA3 being primarily responsible for the de-toxification of the AFB1-epoxide (Ilic et al., 2010; Kensler et al., 2014). The initial period of sensitivity of neonatal mice to AFB1is rapidly overcome via increasing the expression of GSTA3 in the postnatal period (Ilic et al., 2010;Shupe and Sell, 2004).
Pregnancy alters the expression of metabolic enzymes, in-cluding cytochrome P450s and GSTs, that process endogenous
and exogenous chemicals (Koh et al., 2011; Wen et al., 2013;
Zhang et al., 2008). These metabolic changes may stem from pregnancy-induced alterations in the levels of female sex hor-mones such as estradiol, progesterone, and placental growth hormone (Selevan et al., 2000;Zhang et al., 2008). Therefore, in principle, pregnancy-associated metabolic changes could en-hance bioactivation, decrease detoxification of xenobiotics, and thus compromise maternal health. To test that hypothesis, the present study evaluated the effects of pregnancy as a susceptibil-ity factor to the toxicological impact of AFB1 exposure. We dis-covered that following single-dose AFB1 exposure, pregnant females demonstrated higher levels of AFB1-epoxide damage to DNA. The increased levels of DNA damage define pregnancy as a critical window of susceptibility to an important environmental carcinogen. These data also open the possibility that the more susceptible pregnant female may enable greater than expected levels of AFB1exposure to the fetus, potentially increasing its life-long risk of developing cancer due to early life exposure.
MATERIALS AND METHODS
Chemicals. AFB1 (98% purity) and dimethylsulfoxide (DMSO; 99.9%) were purchased from Sigma-Aldrich Corp. (St Louis, Missouri). All other chemicals were at least of ACS reagent grade and obtained from commercial suppliers.
Animals. Female C57BL/6 gpt delta transgenic mice (a gift from Dr Nohmi et al., 1996) were mated with C3H/HeJ males pur-chased from the Jackson Laboratories (Bar Harbor, Maine). GPT mice were utilized in order to maintain congruency with previ-ous studies within the lab, as well as enable future mutagenesis assays. Pregnant mice (n ¼ 8) were administered AFB1(6 mg/kg dissolved in 100 ll DMSO) on GD14 via IP injection. Age-matched nonpregnant C57BL/6 gpt delta mice were treated identically. Six hours after AFB1administration, mice were sacrificed via CO2inhalation; their livers were surgically extracted, snap fro-zen and stored at 80C. Liver tissue samples from AFB1-treated animals were subsequently used for DNA adduct analy-sis. Liver tissue samples from age-matched groups of pregnant and nonpregnant animals that were not treated with AFB1were harvested similarly and used for transcript and protein analysis.
Figure 1. Overview of AFB1metabolism. AFB1is metabolically activated by phase
I enzymes, including cytochromes P450 CYP1A2 or CYP3A family members to the reactive AFB1epoxide, which can damage DNA by forming the AFB1-N7
-gua-nine covalent adduct. The main pathway to detoxify the epoxide involves the phase II enzymes GSTs, which chemically inactivate it by conjugation to GSH (Kensler et al., 2003).
All experiments were conducted in accordance with protocols approved by the MIT Committee on Animal Care.
DNA adduct analysis in liver tissues. DNA was isolated from liver tissue samples (n ¼ 8) using previously described methods (Woo et al., 2011). AFB1-DNA adducts were released from DNA by hydro-lysis in 1.0 N HCl at 95C for 15 min (Groopman et al., 1981).15 N5-Guanine-derived AFB1-adduct internal standards were added to the hydrolytically released AFB1-N7-guanine adducts to permit quantitative analysis by isotope dilution mass spectrometry. Prior to analysis by mass spectrometry, AFB1-DNA adduct mixtures were separated by ultra-high performance liquid chromatography (UPLC). The protonated parent ion of the AFB1-N7-guanine adduct (m/z 480.1) was selected and subjected to collision-induced frag-mentation producing a m/z 152 product ion that was monitored to quantify adduct levels (Egner et al., 2006).
Western blot analysis. Liver tissue (n ¼ 4) was homogenized in radio-immunoprecipitation assay buffer (RIPA, Santa Cruz Biotechnology, Dallas Texas), which was supplemented with protease inhibitors (phenylmethylsulfonyl fluoride (PMSF; 10 mM) and Leupeptin, (0.01 mg/ml). Laemmli buffer was added, followed by denaturation via boiling for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and trans-ferred to an Immobilon-P PVDF membrane (Millipore, Billerica, Massachusetts). Membranes were blocked with 5% BSA (Jackson Immuno-research, West Grove, Pennsylvania) in a Tris-buffered saline solution containing 0.1% Tween 20 followed by treatment with a primary antibody diluted in blocking solution. AntiGSTA3 was a kind gift from Dr John D. Hayes, University of Dundee, UK. AntiGSTA1/2 (No. ABS1651) was from Millipore, AntiCYP3A4 (No. 18227 1 AP0) was from Proteintech (Rosemont, Illinois). AntiCYP1A2 (No. AP11325c) was purchased from Abgent (San Diego, California). Antib-Actin (sc No. 69879) was from Santa Cruz Biotechnology, and antiGAPDH (No. NB300-221) was from Novus Biologicals (Littleton, Colorado). Immune com-plexes were allowed to react with the appropriate secondary antibodies conjugated to horseradish peroxidase (Cell Signaling No. 7074 S, Danvers, Massachusetts) and were visualized with enhanced chemiluminescence (Cell Signaling No. 6883P3). Quantitative real-time PCR. Total RNA was extracted from snap frozen liver samples from untreated pregnant and non-pregnant mice using Tri reagent (Sigma Aldrich) (n ¼ 6). RNA was purified using the RNeasy Mini Kit and treated with DNase to remove any potential genomic DNA contamination (Qiagen, Germantown, Maryland). RNA quantity and quality was assessed via spectro-photometric measurement at A260nmand A260nm/A280nm. cDNA was synthesized from 1 mg of RNA using the Quantitect reverse transcription kit (Qiagen). Primer sequences (see Supplementary Table 1) were obtained from PrimerBank or custom designed and ordered from Integrated DNA technologies (Coralville, Iowa) (Spandidos et al., 2010). qRT-PCR reactions were run in quadrupli-cate utilizing Quantitect SYBR green (Qiagen) on a Roche Light Cycler 480. The purity of PCR products was assessed by melting temperature and size determined by agarose gel. PCR efficiency was calculated by reference to a standard curve using serial sam-ple dilutions. Relative transcript levels were determined using thePfaffl (2001) method.
Statistical analyses. Student’s 2-tailed t tests were used to deter-mine the significance of differences in DNA adduct levels, Western analyses of proteins and mRNA levels assayed by RT-PCR between experimental groups.
RESULTS
AFB1-DNA Adduct Formation Is Enhanced During Pregnancy We used the AFB1-N7-guanine adduct as a biomarker to investi-gate the efficiency of both phase I and II metabolism of AFB1in pregnant and nonpregnant mice (Figure 1). Six hours post AFB1 administration, mice were sacrificed and DNA was isolated from liver tissue. Subsequently, the AFB1-N7-guanine adduct levels were analyzed by quantitative isotope dilution mass spectrometry (Egner et al., 2006). We found that AFB1-N7 -gua-nine adduct levels were approximately 2-fold greater in the liv-ers of GD14 pregnant mice compared with adduct levels in nonpregnant mice (19.2 6 4.22 pmol adduct/mg DNA vs 8.61 6 0.70 pmol adduct/mg DNA; p ¼ .027;Figure 2).
Pregnancy Modulates the Expression of AFB1Metabolizing Enzymes in a Manner That Favors Toxicity
Following the observation of a 2-fold increase in AFB1-N7 -gua-nine adducts in the livers of pregnant over nonpregnant mice, we examined the gene expression of key metabolic enzymes in the aflatoxin activation and detoxification pathways. In untreated animals, phase I proteins that have been shown to be responsible for the bioactivation of AFB1were found to have dif-ferentially increased expression in pregnant versus nonpreg-nant mice. Specifically, murine CYP(s) recognized by the antibody to human CYP3A4 showed a approximately 3-fold in-crease, and CYP1A2 showed a 5-fold increase (Figure 3). In our experiments below, we cannot be certain that the human anti-body used detects specifically the corresponding mouse homo-log; hence, we consider the murine response to a given human antibody (eg, human CYP3A4 or CYP1A2) to be taken with the caveat that the antibody may show unexpected crossreactivity. Given this operational definition, the observed upregulation of these CYPs, CYP3A4, and CYP1A2, may be responsible for the in-creased AFB1-N7-guanine adduct burden in pregnant mice. To further probe the regulation of CYPs during pregnancy we deter-mined transcript levels via RT-PCR. No change was detected in the transcript levels of Cyp1a2 between the pregnant and non-pregnant mice (Figure 4). Because of uncertainty in the specific-ity of the human 3A4 antibody for mouse CYP3A family proteins, we performed a Protein Blast that identified 8 mouse CYPs having >70% homology to human CYP3A4. These CYP3A4 homologs were probed via RT-PCR, with Cyp3a57 showing a sig-nificant increase, and Cyp3a16 and Cyp3a44 showing trends of increased transcript expression in pregnant mice (Figure 4).
Figure 2. Pregnancy confers an elevated risk of AFB1-induced DNA damage.
AFB1-N7-Gua levels in liver DNA from nonpregnant (n ¼ 8) and C57BL/6 J
preg-nant mice (n ¼ 8). Mice treated with 6 mg kg1of AFB
1were sacrificed 6 h later
GSTA3, the primary enzyme responsible for phase II metabo-lism of AFB1in mice (Kensler et al., 2014;Van Ness et al., 1998), showed no significant change in expression between pregnant and nonpregnant animals at either protein (Figure 3) or tran-script levels (Figure 3). However, we observed a 2-fold reduction in the protein levels of GSTA1/2, (Figure 3), with a concurrent re-duction in transcript levels (Figure 4). This reduction in GSTA1/2
is consistent with a compromised ability to mitigate oxidative and other stresses, and could increase the half-life of the geno-toxic AFB1epoxide.
DISCUSSION
Our study is consistent with the view that pregnancy can in-crease aflatoxin genotoxicity (inin-creased AFB1DNA adducts) by enhancing the metabolic activation of the toxin (Figure 5). In support of this notion, we found that pregnancy, specifically at GD14, was associated with significantly elevated AFB1 DNA adducts 6 h post-exposure. GD14 of pregnancy also demon-strated increased protein levels of the phase I metabolism enzymes CYP1A2 and mouse homologs of human CYP3A4, which are known to bioactivate AFB1. Additionally, pregnancy did not affect the levels of GSTA3, the native powerful counter-measure to the AFB1epoxide. As a result, it is possible that the increase in metabolic activation afforded by the phase I enzymes shifts the equilibrium of AFB1 activation/detoxifica-tion in favor of activaactivation/detoxifica-tion, yielding an increase in the levels of DNA adducts. Moreover, the reduction in the protein levels of GSTA1/2 may further increase stress on the GSH peroxidase pathway, yielding a reduction in the intracellular reduced GSH pool. In turn, the decreased levels of reduced GSH may hinder the ability of GSTA3 to rapidly detoxify the AFB1 epoxide. Furthermore, diminished levels of GSTA1/2 could lead to greater susceptibility to other toxicological insults, including oxidative stress, altering cellular homeostasis to the detriment of efficient AFB1detoxification. More research, however, is needed to fully understand the implications of decreased GSTA1/2 levels on the toxicological impact of AFB1.
Figure 3. Pregnancy yields an increase in expression of bioactivating enzymes of AFB1. Western blot analysis of representative phases I and II metabolic enzymes in
untreated nonpregnant and GD14 pregnant mice (biological replicates shown for each condition). Fold change was measured via densitometry. Relative protein levels were normalized to the levels of b-actin and GAPDH. *denoted p < .05 (mean 6 SEM; n ¼ 4).
Figure 4. Pregnancy alters expression of Cyp450 genes in the mouse. A host of phase I and II enzyme expression was analyzed via RT-PCR. A significant reduc-tion in expression is present in GSTA1 (p < .05), with trends of increased expres-sion for both Cyp3A13 and Cyp3A44. Relative transcript expresexpres-sion was normalized to GAPDH levels. *denoted p < .05 Data shown are mean 6 SEM; n ¼ 6.
AFB1causes mutations in both male and female mice when administered either prenatally or in the early life stage. This ex-posure, however, is better tolerated in female mice, as demon-strated by lower rates of cancer incidence later in life when compared with males. Both sexes are resistant to the effects of AFB1after reaching adulthood, withstanding doses of up to 60 mg/kg (Vesselinovitch et al., 1972;Wogan, 1969). This effect is due to the induction of expression of GSTA3 approximately one week after birth (Shupe and Sell, 2004). Similarly to aflatoxin, the DNA ethylating agent N-nitrosodiethylamine is a potent in-ducer of HCC in mice, with a sex bias that disproportionately favors female survival (Nakatani et al., 2001). Studies from the DEN literature show that estrogens inhibit production of inflam-matory cytokines, such as interleukin-6 (IL-6) from Kupffer cells. Hence, in estrogen-producing females, a reduction in IL-6 decreases the inflammatory response in the liver following toxin exposure and thus could reduce liver cancer risk (Naugler et al., 2007). By contrast, male mice have lower estrogen levels, which translates to high levels of IL-6, which stimulates inflam-mation and leads to cytotoxicity as evidenced by elevated se-rum aminotransferases and apoptosis (Naugler et al., 2007). The proinflammatory environment acts as a tumor promoter, stim-ulating hepatocyte proliferation and leading to an increased in-cidence of HCC (Naugler et al., 2007). This phenotype may be mirrored in male mice exposed to aflatoxin early in life.
This work did not directly investigate the mechanistic con-nection between the pregnancy-related steroid hormones such as b-estradiol and progesterone and the genotoxic effects of aflatoxin but our results suggest that such studies represent a logical next step. The modulatory effects of pregnancy on meta-bolic enzymes have been examined previously. Wen and co-workers have demonstrated that pregnancy leads to altera-tions in phase II metabolism, specifically reducaltera-tions in Uridine 5’-diphospho-glucuronosyltransferases at both the transcript and protein levels, as well as reductions in GSTA1 and GSTA4 transcripts at GD14 (Wen et al., 2013). Contrary to our results, they did not observe a reduction in GSTA1 protein levels.
Pregnancy-related hormones can act as transcriptional regulators of cytochrome P450 expression (Choi et al., 2013).
In primary human hepatocyte cultures, estradiol enhances the expression of CYP3A4, CYP2A6, and CYP2B6, while progesterone induces the expression of CYP3A4, CYP2A6, CYP2B6, CYP2C8, and CYP3A5 (Choi et al., 2013). In mice, estradiol and glucocorti-coid hormones can potentiate the effects of growth hormones on induction of homologs of CYP3A4 (CYP3A41 and CYP3A11) expression (Sakuma et al., 2004). In fact, increased protein ex-pression and activity of CYP3A family members during preg-nancy has been reported by several studies, with concurrent increased transcripts of Cyp3a16, Cyp3a41, and Cyp3a44 (Isoherranen and Thummel, 2013;Zhang et al., 2008). With re-gard to CYP1A2, the literature precedent is less definitive. One study, in which rats were exposed to estradiol, reported eleva-tion of CYP1A2 expression, which is in agreement with the pre-sent study, and enhancement in the expression of CYP2C (CYP2C6, CYP2C7, and CYP2C12) (Choi et al., 2011). Other studies, which are at odds with ours, show that the hormones produced during pregnancy, including estrogens, progesterone and growth hormone, reduce the levels of CYP1A2 (Isoherranen and Thummel, 2013;Koh et al., 2011). Further work is needed to rec-oncile these divergent observations.
Pregnancy-dependent increased aflatoxin burden on the mother may have downstream consequences to fetal health and development. Studies have demonstrated aflatoxin as a transplacental xenobiotic both in mice (Chawanthayatham et al., 2015) and in humans (Abdulrazzaq et al., 2002;Denning et al., 1990;Hsieh and Hsieh, 1993). Although the exact method of bioactivation and fetal transmission is not fully elucidated, the developing fetus represents a critical window of susceptibil-ity to AFB1. Our lab has demonstrated that in utero exposure to AFB1 leads to an increase in mutations (Chawanthayatham et al., 2015), and others have shown that humans exposed to AFB1in utero leads to reduced birthweight (Abdulrazzaq et al., 2002;Turner et al., 2007), alteration of methylation profiles of white blood cells (Hernandez-Vargas et al., 2015) and other effects.
In conclusion, our study establishes pregnancy as a risk fac-tor for AFB1genotoxicity; 6 h post-exposure to the toxin, the pregnant mice accumulate twice as many AFB1-N7-guanine adducts as the nonpregnant control group. This result may be partially explained by the increased levels of phase I metaboliz-ing enzymes in pregnant animals, coupled with the lack of a compensatory change in the phase II enzymes (Figure 3). Further lines of research into the effects of chronic AFB1 expo-sure throughout pregnancy may reveal significantly greater im-pact on both maternal and fetal health.
Perhaps of greater importance is the revelation of pregnancy as a generalizable risk factor for toxicological impact. The ob-served reduction in the expression of a representative phase II enzyme, GSTA1/2, in pregnant animals versus nonpregnant controls suggests that pregnancy may confer increased risk to a host of endogenous and exogenous toxicants, in addition to AFB1. The genotoxic vulnerability conferred by pregnancy emphasizes the need not only to protect susceptible prenatal life, but also to lessen the exposure of the mother to environ-mental chemicals. Indeed, the knowledge of the need and subsequent interventions to protect a more vulnerable mother may reasonably offer the secondary benefit of protection to the fetus.
SUPPLEMENTARY DATA
Supplementary data are available at Toxicological Sciences online.
Figure 5. Pregnancy enhances the toxicological impact of AFB1. The observed
increase in phase I metabolic enzymes (CYP1A2, CYP3A) in pregnant mice leads to increased levels of AFB1-epoxide, while amounts of the phase II detoxifying
enzyme (GSTA3) remain unchanged. This effect results in enhanced genotoxic stress as measured by the levels of AFB1-N7-Gua adducts, a biomarker of AFB1
FUNDING
This work was supported by the National Institutes of Health (T32-ES007020, P30-ES002109, R01-ES016313, R01-CA080024, P30-CA006973).
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